1. Field of the Invention
This invention relates to reagents and methods for facilitating the entry of biologically-active compounds into phagocytic cells. The invention specifically provides particulate carriers generally termed microparticles comprising antimicrobial compounds, both per se as compositions of matter and as pharmaceutical compositions thereof. Alternative embodiments of said microparticle carriers are provided wherein one or a multiplicity of antimicrobial compounds are linked to a microparticle via a specifically-cleaved linker moiety, or wherein a porous microparticle is impregnated with one or a multiplicity of antimicrobial compounds, or wherein the microparticle is coated with one or a multiplicity of antimicrobial compounds, wherein the impregnated or coated microparticle is further coated with a specifically-degradable coating material, wherein in their respective embodiments the specifically-cleaved linker moiety and the specifically-degradable coating material are the targets of a microorganism-specific protein having an enzymatic activity not otherwise expressed in the phagocytic cell, or that is specifically expressed by the phagocytic cell only when infected with said microorganism. Thus, the invention provides cell targeting of drugs to phagocytic cells wherein the targeted drug is only released in phagocytic cells that infected with a particular microorganism. Methods of treating diseases having an intracellular microbial etiology are also provided, particularly for the treatment of tuberculosis and other Mycobacterium-caused diseases.
2. Background of the Related Art
A major goal in the pharmacological arts has been the development of reagents and methods for facilitating specific delivery of therapeutic compounds, drugs and other agents to the appropriate cells and tissues that would benefit from such treatment, and the avoidance of the general physiological effects of systemic or otherwise inappropriate delivery of such compounds, drugs or agents to other cells or tissues of the body. The most common example of the need for such specificity is in the field of antibiotic therapy, in which the amount of a variety of antibiotic, antimicrobial and antiviral compounds, drugs and agents that can be safely administered to a patient is limited by their cytotoxic and immunogenic effects.
It is also recognized in the medical arts that certain cells are the sites of pharmacological action of certain compounds, drugs or agents or are involved in the biological response to certain stimuli. In particular, it is now recognized that certain cell types are reservoirs for occult infection that evades normal immune surveillance and permits the persistence of a chronically infected disease state. Specific delivery of diagnostic or therapeutic compounds, drugs or agents to such cells is thus desirable to increase the specificity and effectiveness of clinical diagnostic or therapeutic techniques.
A. Drug Targeting
It is desirable to increase the efficiency and specificity of administration of a therapeutic compound, drug or agent to the cells of the relevant tissues in a variety of pathological states. This is particularly important as relates to antibiotic, antimicrobial and antiviral compounds, drugs or agents. These compounds, drugs or agents typically have pleiotropic antibiotic, immunogenic, cytopathic and cytotoxic effects that damage or destroy uninfected cells as well as infected cells. In addition, certain compounds, drugs or agents are xe2x80x9cactivatedxe2x80x9d or chemically modified by an enzymatic or chemical activity specific for infected cells, in which activated form the compounds, drugs or agents are particularly toxic. Resistance to these types of compounds, drugs or agents can arise by attenuation, mutation or ablation of the chemical or enzymatic activity in the infected cell. Thus, an efficient delivery system which would enable the delivery of such compounds, drugs or agents, particularly said xe2x80x9cactivatedxe2x80x9d forms thereof, specifically to infected cells would increase the efficacy of treatment, overcome drug resistance, reduce the associated xe2x80x9cside effectsxe2x80x9d of such drug treatments, and also serve to reduce morbidity and mortality associated with clinical administration of such compounds, drugs or agents.
Numerous methods for enhancing the cytotoxic activity and the specificity of antibiotic drug action have been proposed. One method, receptor targeting, involves linking the therapeutic agent to a ligand which has an affinity for a receptor expressed on the desired target cell surface. Using this approach, antibiotic, antimicrobial and antiviral compounds, drugs and agents are intended to adhere to the target cell following formation of a ligand-receptor complex on the cell surface. Entry into the cell could then follow as the result of internalization of ligand-receptor complexes. Following internalization, the antibiotic, antimicrobial and antiviral compounds, drugs and agents may then exert therapeutic effects directly on the cell.
The ligand-receptor approach is plagued by a number of biological limitations. Receptor-mediated uptake does not specifically target infected cells; all cells that happen to express the receptor take up the drug. A further limitation of the receptor targeting approach lies in the fact that there are only a finite number of receptors on the surface of target cells. It has been estimated that the maximum number of receptors on a cell is approximately one million (Darnell et al., 1990, Molecular Cell Biology, 2d ed., W. H. Freeman: N.Y.). This estimate predicts that there may be a maximum one million drug-conjugated ligand-receptor complexes on any particular cell. Since not all of the ligand-receptor complexes may be internalized, and any given ligand-receptor system may express many-fold fewer receptors on any particular cell surface, the efficacy of intracellular drug delivery using this approach is uncertain. Other known intracellular ligand-receptor complexes (such as the steroid hormone receptor) express as few as ten thousand hormone molecules per cell, and thus are even less suitable for mediating cell-specific targeting of antibiotic, antibiotic or antiviral compounds, drugs and agents. Id. Finally, once the bound drug entered a cell, it would not be expected to be differentially released in infected cells.
Other methods of delivering therapeutic agents at concentrations higher than those achievable through the receptor targeting process include the use of lipid conjugates that have selective affinities for specific biological membranes. These methods have met with little success (see, for example, Remy et al., 1962, J. Org. Chem. 27: 2491-2500; Mukhergee and Heidelberger, 1962, Cancer Res. 22: 815-22; Brewster et al., 1985, J. Pharm. Sci. 77: 981-985).
Liposomes have been used to attempt cell targeting
U.S. Pat. No. 5,223,263, issued Jun. 29, 1993 to Hostetler et al. disclose conjugates between antiviral nucleoside analogues and polar lipids.
U.S. Pat. No. 5,484,809, issued Jan. 16, 1996 to Hostetler et al. disclose taxol and taxol derivatives conjugated to phospholipids.
U.S. Pat. No. 5,580,571, issued Dec. 3, 1996 to Hostetler et al. disclose nucleoside analogues conjugated to phospholipids.
U.S. Pat. No. 5,744,461, issued Apr. 28, 1998 to Hostetler et al. disclose nucleoside analogues conjugated to phosphonoacetic acid lipid derivatives.
U.S. Pat. No. 5,744,592, issued Apr. 28, 1998 to Hostetler et al. disclose nucleoside analogues conjugated to phospholipids.
U.S. Pat. No. 5,756,116, issued May 26, 1998 to Hostetler et al. disclose nucleoside analogues conjugated to phospholipids.
International Patent Application Publication Number WO89/02733, published April 1989 to Vical disclose conjugates between antiviral nucleoside analogues and polar lipids.
European Patent Application Publication Number 0350287A2 to Vical disclose conjugates between antiviral nucleoside analogues and polar lipids.
International Patent Application Publication Number WO93/00910 to Vical disclose conjugates between antiviral nucleoside analogues and polar lipids.
Rahman et al., 1982, Life Sci. 31: 2061-71 found that liposomes which contained galactolipid as part of the lipid appeared to have a higher affinity for parenchymal cells than liposomes which lacked galactolipid.
Gregoriadis, 1995, Trends in Biotechnology 13: 527-537 reviews the xe2x80x9cprogress and problemsxe2x80x9d associated with using liposomes for targeted drug delivery.
Ledley, 1995, Human Gene Therapy 6: 1129-1144 reviews the use of liposomes for gene therapy.
Mickisch, 1995, World J. Urology 13: 178-185 reviews the use of liposomes for gene therapy of renal cell carcinoma.
Yang et al. 1997, J. Neurotrauma 14: 281-297 review the use of cationic liposomes for gene therapy directed to the central nervous system.
Storm and Crommelin, 1997, Hybridoma 16: 119-125 review the preliminary use of liposomes for targeting chemotherapeutic drugs to tumor sites.
Manusama et al., 1998, Semin. Surg. Oncol. 14: 232-237 reported on preclinical and clinical trials of liposome-encapsulated tumor necrosis factor for cancer treatments.
To date, however, efficient or specific drug delivery has not been predictably achieved using drug-encapsulated liposomes.
Drug delivery to specific sites or cells has been attempted as a way to enhance drug effectiveness. In one example of this approach, prodrug activation has been attempted using antibodies to provide xe2x80x9ctime-releasedxe2x80x9d drug delivery agents (Bignami et al., 1992, Cancer Res. 52: 5759-5764). In this approach, a specific targeting antibody conjugated with a prodrug-activating enzyme was used to activate a systemically-delivered prodrug only at the specific site recognized by the antibody.
There remains a need for the development of cell-specific drug targeting and delivery systems, particularly with antibiotic, antimicrobial and antiviral compounds, drugs and agents.
B. Phagocytic Cell-Specific Targeting
Cell-specific targeting is an important goal of antimicrobial therapy, particularly in the event that a specific cell type is a target of acute or chronic infection. Targeting a specific infected cell type would be advantageous because it would allow administration of antibiotic, antimicrobial or antiviral compounds, drugs or agents to an animal suffering from infection with a microbial pathogen, without the risk of non-specific toxicity to uninfected cells that would exist with nontargeted administration of toxic compounds, and because it would permit administration of dosages unattainable using systemically-administered, non-targeted embodiments of such antibiotic, antiviral and antimicrobial compounds, drugs and agents. This is particularly true of xe2x80x9cactivatedxe2x80x9d compounds, drugs or agents, which are by definition particularly toxic forms of said compounds, drugs or agents and particularly efficient in their antibiotic, antimicrobial, or antiviral properties. An additional advantage of such targeted antimicrobial therapy would be improved pharmacokinetics that would result from specific concentration of antibiotic, antimicrobial or antiviral compounds, drugs and agents to the infected cells that are the sites of infection.
Phagocytic cells such as monocytes and macrophages are known to be specific targets for infection of certain pathogenic microorganisms.
Sturgill-Koszycki et al., 1994, Science 263: 678-681 disclose that the basis for lack of acidification of phagosomes in M. avium and M. tuberculosis-infected macrophages is exclusion of the vesicular proton-ATPase.
Sierra-Honigman et al., 1993, J. Neuroimmunol. 45: 31-36 disclose Borna disease virus infection of monocytic cells in bone marrow.
Maciejewski et al., 1993, Virol. 195: 327-336 disclose human cytomegalovirus infection of mononucleated phagocytes in vitro.
Alvarez-Dominguez et al., 1993, Infect. Immun. 61: 3664-3672 disclose the involvement of complement factor C1q in phagocytosis of Listeria monocytogenes by macrophages.
Kanno et al., 1993, J. Virol. 67: 2075-2082 disclose that Aleutian mink disease parvovirus replication depends on differentiation state of the infected macrophage.
Embretson et al., 1993, Nature 362: 359-362 disclose covert infection of macrophages by human immunodeficiency virus.
Meltzer and Gendelman, 1992, Curr. Top. Microbiol. Immunol. 181: 239-263 disclose infection of mononuclear phagocytes with human immunodeficiency virus.
Kanno et al., 1992, J. Virol. 66: 5305-5312 disclose that Aleutian mink disease parvovirus infects peritoneal macrophages in mink.
Narayan et al., 1992, J. Rheumatol. 32: 25-32 disclose arthritis in animals caused by infection of macrophage precursors with lentivirus, and activation of quiescent lentivirus infection upon differentiation of such precursor cells into terminally-differentiated macrophages.
Horwitz, 1992, Curr. Top. Microbiol. Immunol. 181: 265-282 disclose Legionella pneumophila infections of alveolar macrophages as the basis for Legionnaire""s disease and Pontiac fever.
Sellon et al., 1992, J. Virol. 66: 5906-5913 disclose that equine infectious anemia virus replicates in tissue macrophages in vivo.
Groisman et al., 1992, Proc. Natl. Acad. Sci. USA 89: 11939-11943 disclose that S. typhimurium survives inside infected macrophages by resistance to antibacterial peptides.
Friedman et al., 1992, Infect. Immun. 60: 4578-4585 disclose Bordetella pertussis infection of human macrophages.
Stellrecht-Broomhall, 1991, Viral Immunol. 4: 269-280 disclose that lymphocytic choriomeningitis virus infection of macrophages promotes severe anemia caused by macrophage phagocytosis of red blood cells.
Frehel et al., 1991, Infect. Immun. 59: 2207-2214 disclose infection of spleen and liver-specific inflammatory macrophages by Mycobacterium avium, the existence of the microbe in encapsulated phagosomes within the inflammatory macrophages and survival therein in phagolysosomes.
Bromberg et al., 1991, Infect. Immun. 59: 4715-4719 disclose intracellular infection of alveolar macrophages.
Mauel, 1990, J. Leukocyte Biol. 47: 187-193 disclose that Leishmania spp. are intracellular parasites in macrophages.
Buchmeier and Heffron, 1990, Science 248: 730-732 disclose that Salmonella typhimurium infection of macrophages induced bacterial stress proteins.
Panuska et al., 1990, J. Clin. Invest. 86: 113-119 disclose productive infection of alveolar macrophages by respiratory syncytial virus.
Cordier et al., 1990, Clin. Immunol. Immunopathol. 55: 355-367 disclose infection of alveolar macrophages by visna-maedi virus in chronic interstitial lung disease in sheep.
Schlessinger and Horwitz, 1990, J. Clin. Invest. 85: 1304-1314 disclose Mycobacterium leprae infection of macrophages.
Schmidt et al., 1990, Res. Virol. 141: 143-152 disclose infection of primary cultures of liver Kupffer cells with human immunodeficiency virus.
Clarke et al., 1990, AIDS 4: 1133-1136 disclose human immunodeficiency virus infection of alveolar macrophages in lung.
Baroni et al., 1988, Am. J. Pathol. 133: 498-506 disclose human immunodeficiency virus infection of lymph nodes.
Payne et al, 1987, J. Exp. Med. 166: 1377-1389 disclose Mycobacterium tuberculosis infection of macrophages.
Murray et al., 1987, J. Immunol. 138: 2290-2296 disclose that liver Kupffer cells are the initial targets for L. donovani infection.
Koenig et al., 1986, Science 233: 1089-1093 disclose human immunodeficiency virus infection of macrophages in the central nervous system.
Gendelman et al., 1985, Proc. Natl. Acad. Sci. USA 82: 7086-7090 disclose infection of phagocytic cells with lentivirus.
Horwitz and Maxfield, 1984, J. Cell Biol. 99: 1936-1943 disclose that L. pneumophila survives in infected phagocytic cells at least in part by inhibiting reduction of intraphagosomic hydrogen ion concentration (pH).
Shanley and Pesanti, 1983, Infect. Immunol. 41: 1352-1359 disclose cytomegalovirus infection of macrophages in murine cells.
Horwitz, 1983, J. Exp. Med. 158: 2108-2126 disclose that L. pneumophila is an obligate intracellular parasite that is phagocytized into a phagosome wherein fusion with lysosome is inhibited.
Chang, 1979, Exp. Parasitol. 48: 175-189 disclose Leischmania donovani infection of macrophages.
Wyrick and Brownridge, 1978, Infect. Immunol. 19: 1054-1060 disclose Chlamydia psittaci infection of macrophages.
Halstead et al., 1977, J. Exp. Med. 146: 201-217 disclosed infection of phagocytic cells with dengue virus.
Nogueira and Cohn, 1976, J. Exp. Med. 143: 1402-1420 disclose Trypanosoma cruzi infection of macrophages.
Jones and Hirsch, 1972, J. Exp. Med. 136: 1173-1194 disclose Toxoplasma gondii infection of macrophages.
Persistent infection of phagocytic cells has been reported in the prior art.
Embretson et al., 1993, Nature 362: 359-361 disclose covert infection of macrophages with HIV and dissemination of infected cells throughout the immune system early in the course of disease.
Schnorr et al., 1993, J. Virol. 67: 4760-4768 disclose measles virus persistent infection in vitro in a human monocytic cell line.
Meltzer and Gendelman, 1992, Curr. Topics Microbiol. Immunol. 181: 239-263 provide a review of HIV infection of tissue macrophages in brain, liver, lung, skin, lymph nodes, and bone marrow, and involvement of macrophage infection in AIDS pathology.
Blight et al., 1992, Liver 12: 286-289 disclose persistent infection of liver macrophages (Kuppfer cells) by hepatitis C virus.
McEntee et al., 1991, J. gen. Virol. 72: 317-324 disclose persistent infection of macrophages by HIV resulting in destruction of T lymphocytes by fusion with infected macrophages, and that the macrophages survive fusion to kill other T lymphocytes.
Kondo et al., 1991, J. gen. Virol. 72: 1401-1408 disclose latent infection by herpes simplex virus 6 of monocytes activated by differentiation into macrophages.
King et al., 1990, J. Virol. 64: 5611-5616 disclose persistent infection of macrophages with lymphocytic choriomeningitis virus.
Schmitt et al., 1990, Res. Virol. 141: 143-152 disclose a role for HIV infection of Kupffer cells as reservoirs for HIV infection.
Gendelman et al., 1985, Proc. Natl. Acad. Sci. USA 82: 7086-7090 disclose lentiviral (visna-maedi) infection of bone marrow precursors of peripheral blood monocytes/macrophages that provide a reservoir of latently-infected cells.
Halstead et al., 1977, J. Exp. Med. 146: 201-217 disclose that macrophages are targets of persistent infection with dengue virus.
Mauel et al., 1973, Nature New Biol. 244: 93-94 disclose that lysis of infected macrophages with sodium dodecyl sulfate could release live microbes.
Attempts at cell-specific drug targeting have been reported in the prior art.
Rubinstein et al., 1993, Pharm. Res. 10: 258-263 report colon targeting using calcium pectinate (CaPec)-conjugated drugs, based on degradation of CaPec by colon specific (i.e., microflora-specific) enzymes and a hydrophobic drug incorporated into the insoluble CaPec matrices.
Sintov et al., 1993, Biomaterials 14: 483-490 report colon-specific targeting using conjugation of drug to insoluble synthetic polymer using disaccharide cleaved by enzymes made by intestinal microflora, specifically, xcex2-glycosidic linkages comprising dextran.
Franssen et al., 1992, J. Med. Chem. 35: 1246-1259 report renal cell/kidney drug targeting using low molecular weight proteins (LMWP) as carriers, using enzymatic/chemical hydrolysis of a spacer molecule linking the drug and LMWP carrier.
Bai et al., 1992, J. Pharm. Sci. 81: 113-116 report intestinal cell targeting using a peptide carrier-drug system wherein the conjugate is cleaved by an intestine-specific enzyme, prolidase.
Gaspar et al., 1992, Ann. Trop. Med. Parasitol. 86: 41-49 disclose primaquine-loaded polyisohexylcyanoacrylate nanoparticles used to target Leschmania donovani infected macrophage-like cells in vitro.
Pardridge, 1992, NIDA Res. Monograph 120: 153-168 report opioid-conjugated chimeric peptide carriers for targeting to brain across the blood-brain barrier.
Bai and Amidon, 1992, Pharm. Res. 9: 969-978 report peptide-drug conjugates for oral delivery and intestinal mucosal targeting of drugs.
Ashborn et al., 1991, J. Infect. Dis. 163: 703-709 disclose the use of CD4-conjugated Pseudomonas aeruginosa exotoxin A to kill HIV-infected macrophages.
Larsen et al., 1991, Acta Pharm. Nord. 3: 41-44 report enzyme-mediated release of drug from dextrin-drug conjugates by microflora-specific enzymes for colon targeting.
Faulk et al., 1991, Biochem. Int. 25: 815-822 report adriamycin-transferrin conjugates for tumor cell growth inhibition in vitro.
Zhang and McCormick, 1991, Proc. Natl. Acad. Sci. USA 88: 10407-10410 report renal cell targeting using vitamin B6-drug conjugates.
Blum et al., 1982, Int. J. Pharm. 12: 135-146 report polystyrene microspheres for specific delivery of compounds to liver and lung.
Trouet et al., 1982, Proc. Natl. Acad. Sci. USA 79: 626-629 report that daunorubicin-conjugated to proteins was cleaved by lysosomal hydrolases in vivo and in vitro.
Shen et al., 1981, Biochem. Biophys. Res. Commun. 102: 1048-1052 report pH-labile N-cis-acontinyl spacer moieties.
Monoclonal antibodies have been used in the prior art for drug targeting.
Serino et al, U.S. Pat. No. 4,793,986, issued Dec. 27, 1988, provides platinum anticancer drugs conjugated to polysaccharide (dextrin) carrier for conjugation to monoclonal antibodies for tumor cell targeting.
Bickel et al., 1993, Proc. Natl. Acad. Sci. USA 90: 2618-2622 discloses the use of a chimeric protein vector for targeting across blood-brain barrier using an anti-transferrin monoclonal antibody.
Rowlinson-Busza and Epenetos, 1992, Curr. Opin. Oncol. 4: 1142-1148 provides antitumor immunotargeting using toxin-antibody conjugates.
Blakey, 1992, Acta Oncol. 31: 91-97 provides a review of antitumor antibody targeting of antineoplastic drugs.
Senter et al., 1991, in Imunobiology of Peptides and Proteins, Vol. VI, pp. 97-105 discloses monoclonal antibodies linked to alkaline phosphatase or penicillin-V amidase to activate prodrugs specifically at site of antibody targeting, for therapeutic treatment of solid tumors.
Drug-carrier conjugates have been used in the prior art to provide time-release drug delivery agents.
Couveur and Puisieux, 1993, Adv. Drug Deliv. Rev. 10: 141-162 provide a review of microcapsule (vesicular), microsphere (dispersed matrix) and microparticle (1-250 xcexcm)-based drug delivery systems, based on degradation of particle with drug release, to provide time release of drugs, oral delivery via transit through the intestinal mucosa and delivery to Kupffer cells of liver.
Duncan, 1992, Anticancer Drugs 3: 175-210 provide a review of improved pharmacokinetic profile of in vivo drug release of anticancer drugs using drug-polymer conjugates.
Heinrich et al., 1991, J. Pharm. Pharmacol. 43: 762-765 disclose poly-lactide-glycolide polymers for slow release of gonadotropin releasing hormone agonists as injectable implants.
Wada et al. 1991, J. Pharm. Pharmacol. 43: 605-608 disclose sustained-release drug conjugates with lactic acid oligomers.
Specifically, polymer-conjugated drugs have been reported in the prior art, and attempts to adapt particulate conjugates have also been reported.
Ryser et al., U.S. Pat. No. 4,847,240, issued Jul. 11, 1989, provides cationic polymers for conjugation to compounds that are poorly transported into cells. Examples include the antineoplastic drug methotrexate conjugated with polylysine and other polycationic amino acids as carriers.
Ellestad et al., U.S. Pat. No. 5,053,394, issued Oct. 1, 1991, provides carrier-drug conjugates of methyltrithiol antibacterial and antitumor agents with a spacer linked to a targeting molecule that is an antibody or fragment thereof, growth factors or steroids.
Kopecek et al., U.S. Pat. No. 5,258,453, issued Nov. 2, 1993, provides antitumor compositions comprising both an anticancer drug and a photoactivatable drug attached to a copolymeric carrier by functional groups labile in cellular lysosomes, optionally containing a targeting moiety that are monoclonal antibodies, hormones, etc.
Yatvin et al., U.S. Pat. No. 5,543,390, issued Aug. 6, 1996, discloses microparticles conjugated to antiproliferative drugs.
Yatvin et al., U.S. Pat. No. 5,543,391, issued Aug. 6, 1996, discloses microparticles conjugated to antiproliferative drugs.
Negre et al., 1992, Antimicrob. Agents and Chemother. 36: 2228-2232 disclose the use of neutral mannose-substituted polylysine conjugates with an anti-leischmanial drug (allopurinol riboside) to treat murine infected macrophages in vitro.
Yatvin, 1991, Select. Cancer. Therapeut. 7: 23-28 discusses the use of particulate carriers for drug targeting.
Hunter et al., 1988, J. Pharm. Pharmacol. 40: 161-165 disclose liposome-mediated delivery of anti-leischmanial drugs to infected murine macrophages in vitro.
Saffran et al., 1986, Science 233: 1081-1084 disclose drug release from a particulate carrier in the gut resulting from degradation of the carrier by enzymes produced by intestinal microflora.
A particular human disease related to infection of phagocytic cells by a microorganism is tuberculosis, caused by infection with Mycobacterium tuberculosis. This disease typically arises after inhalation in phagocytic macrophages in the lung, where characteristic localized sites of infection (termed tubercules) are formed and comprise sites of further systemic infection. Although previously well-controlled by antibiotics such as isoniazid, the development of drug-resistance by the infectious agent, and the increased numbers of immune-compromised individuals with the outbreak of the AIDS crisis has created a near epidemic of tuberculosis cases world-wide. In 1997, the World Health Organization reported tuberculosis to be the world""s top infectious killer.
About one-third of new tuberculosis cases are resistant to the current drug-treatment regimes. It is estimated that drug-resistant tuberculosis accounts for between 2% and 14% of total tuberculosis cases worldwide. As tuberculosis is spread by air-borne droplets from coughing by infected individuals, and its spread is further facilitated in crowded environments such as cities, there is a great potential for a precipitous increase in tuberculosis infections that will not be easily controlled by conventional medicinal intervention such as isoniazid administration. Lethal strains of tuberculosis have the potential for rapid spread, since only about one in ten patients receives the medical treatment necessary to contain and successfully treat the disease. Thus, there exists in this art a need to develop new and better treatments for tuberculosis, particularly tuberculosis infections resistant to traditional antibiotic treatments.
The present invention is directed to improved reagents and methods for delivering antibiotic, antimicrobial or antiviral compounds, drugs or agents to phagocytic cells in vivo and in vitro. In particular, the invention is directed towards delivery of antimicrobial compounds, drugs and agents specific for treatment of tuberculosis and other Mycobacterium-caused diseases in humans.
The invention provides drug delivery vehicles that are microparticles conjugated to, coated with, or impregnated with one or a multiplicity of antimicrobial compounds, drugs or agents specific for the treatment of tuberculosis and other Mycobacterium-caused diseases in animals, most preferably humans. In one preferred embodiment, the antibiotic, antimicrobial or antiviral compound, drug or agent is a prodrug of an activated form of the anti-tuberculosis drug isoniazid. In a second preferred embodiment, the antibiotic, antimicrobial or antiviral compound, drug or agent is a competitive inhibitor of long chain enol-acyl carrier protein reductase (termed InhA), an M. tuberculosis-encoded enzyme required for production of an essential bacterial cell wall component, mycolic acid. In a third preferred embodiment, the antibiotic, antimicrobial or antiviral compound, drug or agent is an irreversible inhibitor of InhA, otherwise termed a xe2x80x9csuicide substratexe2x80x9d herein.
In one aspect, this delivery system achieves specific delivery of antibiotic, antimicrobial or antiviral compounds, drugs or agents to phagocytic cells through conjugating the antibiotic, antimicrobial or antiviral compound, drug or agent with a particular microparticle via a cleavable linker moiety that is specifically cleaved in an infected cell. Alternatively, specific delivery is achieved by impregnating the antibiotic, antimicrobial or antiviral compound, drug or agent into a porous microparticle, which is then coated with a specifically-degraded coating material that is specifically degraded in an infected cell. In yet another alternative embodiment, the delivery system comprises a nonporous microparticle wherein an antibiotic, antiviral and antimicrobial compound, drug or agent is prepared as a coating on the particle surface, and the particle is then further coated by a specifically-degradable coating material that is specifically degraded in an infected cell. In another embodiment, a porous or non-porous microparticle is impregnated or coated with a first antibiotic, antimicrobial or antiviral compound, drug or agent, then coated with a specifically-degradable or non-specifically degradable coating material, then further coated with a second coating of a antibiotic, antimicrobial or antiviral compound, drug or agent that can be the same or different than the first coating of antibiotic, antimicrobial or antiviral compound, drug or agent, then further coated with a second coating of a specifically-degradable or non-specifically degradable coating material that may be the same or different than the first specifically-degradable or non-specifically degradable coating, wherein the microparticle can comprise a multiplicity of such alternating coatings of antibiotic, antimicrobial or antiviral compounds, drugs and agents and specifically-degradable or non-specifically degradable coatings, provided that the final coating of the microparticle is a specifically-degradable coating that is specifically degraded only in a cell infected with a pathological or disease-causing microorganism, most preferably a Mycobacterium species. In each embodiment of the microparticles of the invention, specific release of the antibiotic, antimicrobial or antiviral compounds, drugs and agents from the microparticle is achieved by enzymatic or chemical release of the compound, drug or agent from the microparticle by cleavage of the cleavable linker moiety or the specifically-degradable coating material in infected phagocytic cells. Such microparticles can be produced to provide sequential, delayed, sustained or controlled release of the antibiotic, antimicrobial or antiviral compounds, drugs or agents of the invention.
In a first aspect, the specific delivery of antibiotic, antimicrobial or antiviral compounds, drugs or agents achieved by the present invention results from conjugating, impregnating or coating such compounds, drugs or agents to microparticles. Specific intracellular accumulation and facilitated cell entry is mediated by the phagocytic uptake of microparticle-conjugated antibiotic, antimicrobial or antiviral compounds, drugs or agents by such cells. Preferred embodiments of phagocytic cellular targets include phagocytic hematopoietic cells, preferably macrophages and phagocytic neutrophiles, most preferably macrophages, mononuclear cells and phagocytic neutrophiles from lung tissue.
Particularly preferred targets of the microparticle-conjugated antibiotic, antimicrobial or antiviral compounds, drugs or agents of the invention are phagocytic cells, including phagocytic hematopoietic cells, preferably macrophages and phagocytic neutrophiles and most preferably macrophages, mononuclear cells and phagocytic neutrophiles from lung tissue that are infected with M. tuberculosis, M. africanum, M. bovis or any other microorganism that causes tuberculosis in an animal, most preferably a human. Also preferred targets are cells infected with M. leprae, M. avium, M. intracellulare, M. scrofulaceum, M. kansasii, M. xenopi, M. marinum, M. ulcerans, M. fortuitum and M. chelonae. For such cells, the embodiments of the microparticle-conjugated antibiotic, antimicrobial or antiviral compounds, drugs or agents of the invention are comprised of cleavable linker moieties or specifically-degradable coatings whereby chemical or enzymatic cleavage of said linker moieties or coatings is specific for tuberculosis- or other disease-causing Mycobacterium-infected phagocytic cells. Such microparticles provide for infected cell-specific release of antibiotic, antimicrobial or antiviral compounds, drugs or agents, such as isoniazid, activated isoniazid, rifampin, streptomycin, ethambutol and pyrazinamide, and competitive, non-competitive and xe2x80x9csuicide substratexe2x80x9d InhA inhibitors or any other anti-tuberculosis or anti-Mycobacterium drug or agent, in such infected cells. It is understood that all phagocytic cells are expected to take up such microparticle-conjugated or coated antibiotic, antimicrobial or antiviral embodiments of the invention. However, it is an advantageous feature of the microparticle-conjugated antibiotic, antimicrobial or antiviral compounds of the invention that specific release of biologically-active forms of such antibiotic, antimicrobial or antiviral drugs or agents is dependent on the presence of the infectious microorganism in the phagocytic cell.
The invention provides compositions of matter and pharmaceutical compositions thereof comprising a porous microparticle into which is impregnated with an antibiotic, antimicrobial or antiviral compound, the impregnated porous microparticle being further coated with a specifically-degradable coating material. In this aspect of the invention, the specifically-degradable coating material is specifically degraded inside a phagocytic mammalian cell infected with a tuberculosis-causing or other Mycobacterium-associated disease-causing microorganism, allowing the specific release of the antibiotic, antimicrobial or antiviral compound within the infected cell. In preferred embodiments, the specifically-degradable coating material is a substrate for a protein having an enzymatic activity found specifically in phagocytic cells infected with a tuberculosis-causing or other Mycobacterium-associated disease-causing microorganism. In additional preferred embodiments, the specifically-degraded coating material is chemically cleaved under physiological conditions that are specific for phagocytic cells infected with a tuberculosis-causing microorganism. In preferred embodiments, the antibiotic, antimicrobial or antiviral compound, drug or agent impregnating the microparticle is an activated embodiment of said compound, drug or agent, as defined herein. In alternative embodiments, the microparticle is impregnated with a multiplicity of antibiotic, antimicrobial or antiviral compounds, drugs or agents.
In alternative aspects, the coating material is nonspecifically cleaved chemically or enzymatically inside a phagocytic cell, wherein the antibiotic, antimicrobial or antiviral compound, drug or agent is in a form that is only specifically activated in the cell when the cell is infected with a tuberculosis-causing or other Mycobacterium-associated disease-causing microorganism (wherein said antibiotic, antimicrobial or antiviral compounds, drugs or agents are termed xe2x80x9cprodrugsxe2x80x9d as defined herein when provided in this form). In alternative embodiments, the microparticle is impregnated with a multiplicity of antibiotic, antimicrobial or antiviral compounds, drugs or agents or prodrug embodiments thereof
In preferred embodiments of the invention, the antibiotic compound is a specifically bactericidal or bacteriostatic against a microorganism that causes tuberculosis in an animal, most preferably a human, most preferably M. tuberculosis, M. africanum, M. bovis. Preferred antibiotic compounds used to impregnate such porous microparticles include activated isoniazid, rifampin, streptomycin, ethambutol and pyrazinamide, and competitive, uncompetitive, non-competitive and xe2x80x9csuicide substratexe2x80x9d InhA inhibitors or any other anti-tuberculosis or anti-Mycobacterium compound, drug or agent. Activated and prodrug embodiments of these or other antibiotic, antimicrobial or antiviral compounds, drugs or agents are also preferred, and activated embodiments of said drugs are particularly preferred.
In preferred embodiments, the antimycobacterial drugs used in the practice of the invention are xe2x80x9cactivatedxe2x80x9d embodiments (as defined herein) of competitive, uncompetitive, non-competitive and xe2x80x9csuicide substratexe2x80x9d inhibitors of long chain enol-acyl carrier protein reductase (InhA), a Mycobacterium-specific enzyme necessary for the production of mycolic acid, which an essential component of the mycobacterial cell wall. Inhibition of this enzyme by isoniazid is the basis of current anti-tuberculosis treatment modalities, and resistance to isoniazid is the principle form of drug resistance exhibited by mycobacteria. The compounds of the invention overcome resistance by being xe2x80x9cpre-activatedxe2x80x9d, i.e., these compounds do not rely on activation in the mycobacterium-infected cell for activity (unlike compounds do not rely on activation in the mycobacterium-infected cell for activity (unlike isoniazid itself). Thus, it is expected that resistance is less likely to be developed against these drugs. In a preferred embodiment, these compounds have the generic structure: 
wherein X can be C or O; Y can be N or C; R1 and R2 can each be independently an electron pair, H, CH3, CH2xe2x80x94CH3, or O(CH2)3O or together can be xe2x95x90O, xe2x95x90CH2, xe2x80x94CH2xe2x80x94CH2xe2x80x94, xe2x95x90CHxe2x80x94CHxe2x95x90CH2,xe2x95x90CHxe2x80x94COOCH2xe2x80x94CH3, or OCH2.
The invention also provides compositions of matter and pharmaceutical compositions thereof comprising a nonporous microparticle onto which is coated an antibiotic, antimicrobial or antiviral compound, the coated nonporous microparticle being further coated with a specifically-degradable coating material. In this aspect of the invention, the specifically-degradable coating material is specifically degraded inside a phagocytic mammalian cell infected with a tuberculosis-causing or other Mycobacterium-associated disease-causing microorganism, allowing the specific release of the antibiotic, antimicrobial or antiviral compound within the infected cell. In preferred embodiments, the specifically-degradable coating material is a substrate for a protein having an enzymatic activity found specifically in phagocytic cells infected with a tuberculosis-causing or other Mycobacterium-associated disease-causing microorganism. In additional preferred embodiments, the specifically-degraded coating material is chemically cleaved under physiological conditions that are specific for phagocytic cells infected with a tuberculosis-causing microorganism. In preferred embodiments, the antibiotic, antimicrobial or antiviral compound, drug or agent coating the microparticle is an activated embodiment of said compound, drug or agent, as defined herein. In alternative aspects, the coating material is nonspecifically cleaved chemically or enzymatically inside a phagocytic cell, wherein the antibiotic, antimicrobial or antiviral compound, drug or agent is in a form that is only specifically activated in the cell when the cell is infected with a tuberculosis-causing or other Mycobacterium-associated disease-causing microorganism (wherein said antibiotic, antimicrobial or antiviral compound, drug or agent is termed a xe2x80x9cprodrugxe2x80x9d as defined herein when provided in this form). In alternative embodiments, the microparticle is coated with a multiplicity of antibiotic, antimicrobial or antiviral compounds, drugs or agents or prodrug embodiments thereof.
In preferred embodiments of the invention, the antibiotic compound is a specifically bactericidal or bacteriostatic against a microorganism that causes tuberculosis in an animal, most preferably a human, most preferably M. tuberculosis, M. africanum, M. bovis. Preferred antibiotic compounds used to coat such porous microparticles include activated isoniazid, rifampin, streptomycin, ethambutol and pyrazinamide, and competitive, uncompetitive, non-competitive and xe2x80x9csuicide substratexe2x80x9d InhA inhibitors or any other anti-tuberculosis or anti-Mycobacterium compound, drug or agent. Activated and prodrug embodiments of these or other antibiotic, antimicrobial or antiviral compounds, drugs or agents are also preferred, and activated embodiments are particularly preferred. Most preferred embodiments have the generic structure disclosed above
Additional embodiments of the compositions of matter and pharmaceutical compositions thereof comprising the porous and non-porous, impregnated or coated microparticles of the invention are provided wherein the porous or non-porous microparticle is impregnated or coated with a first antibiotic, antimicrobial or antiviral compound, drug or agent, then coated with a specifically-degradable or non-specifically degradable coating material, then further coated with a second coating of a antibiotic, antimicrobial or antiviral compound, drug or agent that can be the same or different than the first coating of antibiotic, antimicrobial or antiviral compound, drug or agent, then further coated with a second coating of a specifically-degradable or non-specifically degradable coating material that may be the same or different than the first specifically-degradable or non-specifically degradable coating, wherein the microparticle can comprise a multiplicity of such alternating coatings of antibiotic, antimicrobial or antiviral compounds, drugs or agents and specifically-degradable or non-specifically degradable coatings, provided that the final coating of the microparticle is a specifically-degradable coating that is specifically degraded in a cell infected with a pathological or disease-causing microorganism, most preferably a Mycobacterium species. Such microparticles can be produced to provide sequential, delayed, sustained or controlled release of the antibiotic, antimicrobial or antiviral compounds, drugs or agents of the invention. In each embodiment of the microparticles of the invention, specific release of the antibiotic, antimicrobial or antiviral compound, drug or agent from the microparticle is achieved by enzymatic or chemical release of the compound, drug or agent from the microparticle by cleavage of the specifically-degradable coating material in infected phagocytic cells. Antibiotic, antimicrobial and antiviral compounds, drugs or agents released by non-specific chemical or enzymatic degradation are advantageously provided in inactive, prodrug forms that are specifically activated in cells infected with pathological or disease-causing microorganism, most preferably a Mycobacterium species. In one alternative embodiment of this aspect of the invention, the xe2x80x9cgatekeeperxe2x80x9d for release of the antibiotic, antimicrobial or antiviral drug, compound or agent coating the microparticle is the ultimate, specifically-degraded coating material, which is only removed from the microparticle in a phagocytic cell infected with a pathological or disease-causing microorganism, most preferably a Mycobacterium species. In preferred embodiments, the antibiotic, antimicrobial or antiviral agent is provided in an activated form as defined herein. In said preferred embodiments, the xe2x80x9cgatekeeperxe2x80x9d specifically-degraded coating material prevents release of physiologically-significant amounts of the activated compound, drug or agent anywhere other than inside an infected phagocytic cell, most preferably a phagocytic cell infected with a pathological or disease-causing microorganism, most preferably a Mycobacterium species.
In an alternative embodiment, each antimicrobial, antibiotic or antiviral drug, compound or agent is provided in the form of a prodrug that is activated only in a phagocytic cell infected with said pathological or disease-causing microorganism, most preferably a Mycobacterium species. In this alternative embodiment, delivery of the antibacterial, antibiotic or antiviral drug, compound or agent in an active form to a phagocytic cell will only occur in such a cell that is infected with a pathological or disease-causing microorganism, most preferably a Mycobacterium species wherein both the specifically-degradable coating and the prodrug are degraded and activated, respectively, by an enzymatic or chemical reaction specific for the infected cell.
In these aspects of the invention, the antibiotic, antimicrobial or antiviral compound, drug or agent will be understood to dissolve from the surface of the microparticle upon enzymatic or chemical degradation of the organic coating material. Release of the antibiotic, antimicrobial or antiviral compound, drug or agent can be accomplished simply be mass action, i.e., whereby the compound dissolves from the surface of the nonporous microparticle into the surrounding cytoplasm within the cell, or leaches or is released from the porous microparticle.
The invention also provides compositions of matter and pharmaceutical compositions thereof comprising an antibiotic, antimicrobial or antiviral compound, drug or agent linked to a microparticle via a cleavable linker moiety. The cleavable linker moieties of the invention comprise two linker functional groups, wherein the cleavable linker moiety has a first end and a second end. The microparticle is attached to the first end of the cleavable linker moiety through a first linker functional group and the antibiotic, antimicrobial or antiviral compound, drug or agent is attached to the second end of the cleavable linker moiety through a second linker functional group. The cleavable linker moieties of the invention are specifically cleaved inside an infected phagocytic mammalian cell, for example, a phagocytic cell infected with a tuberculosis-causing or other Mycobacterium-associated disease-causing microorganism. In preferred embodiments, the cleavable linker moieties of the invention comprise a substrate for a protein having an enzymatic activity found specifically in phagocytic cells infected with a tuberculosis-causing or other Mycobacterium-associated disease-causing microorganism. In a particular embodiment of this aspect of the invention, the cleavable linker moiety is a peptide of formula (amino acid)n, wherein n is an integer between 2 and 100, preferably wherein the peptide comprises a polymer of one or more amino acids. In additional preferred embodiments, the cleavable linker moieties of the invention are moieties that are chemically cleaved under physiological conditions that are specific for phagocytic cells infected with a tuberculosis-causing microorganism. In preferred embodiments, the antibiotic, antimicrobial or antiviral compound, drug or agent impregnating the microparticle is an activated embodiment of said compound, drug or agent, as defined herein. In alternative embodiments, the microparticles of the invention are provided comprising either a multiplicity of antimicrobial, antibiotic or antiviral compounds, drugs, or agents or a multiplicity of cleavable linker moieties, or both.
In alternative aspects, the cleavable linker moieties are nonspecifically cleaved chemically or enzymatically inside a phagocytic cell, wherein the antibiotic, antimicrobial or antiviral compound, drug or agent is in a form that is only specifically activated in the cell when the cell is infected with a tuberculosis-causing or other Mycobacterium-associated disease-causing microorganism (wherein said antibiotic, antimicrobial or antiviral compounds, drugs or agents are termed xe2x80x9cprodrugsxe2x80x9d as defined herein when provided in this form).
In preferred embodiments of the invention, the antibiotic compound is a specifically bactericidal or bacteriostatic against a microorganism that causes tuberculosis in an animal, most preferably a human, most preferably M. tuberculosis, M. africanum, M. bovis. In preferred embodiments, the antibiotic compound is isoniazid, activated isoniazid, rifampin, streptomycin, ethambutol and pyrazinamide, and competitive, uncompetitive, non-competitive and xe2x80x9csuicide substratexe2x80x9d InhA inhibitors or any other anti-tuberculosis or anti-Mycobacterium compound, drug or agent. Activated and prodrug embodiments of these or other antibiotic, antimicrobial or antiviral compounds, drugs or agents are also preferred.
The most preferred embodiments of the microparticles of the invention comprise prodrugs forms of activated isoniazid conjugates with NAD (termed isoniazid-NAD analogues, of INA, herein) that are inactivated by covalent modification of the activated drug to block binding of the drug to NAD-requiring enzymes, including InhA and mammalian cell, most preferably human cell-derived, NAD requiring enzymes. In the most preferred embodiments of this aspect of the invention, the inactivated prodrug form is specifically activated only in Mycobacterium-infected cells. In one aspect, such specific cleavage is due to a chemical linkage in the derivative that is labile within the infected cell due to conditions caused by or that result from infection of the cell with the mycobacteria. In another preferred aspect, such specific cleavage is due to an enzymatic activity which is produced either by the mycobacteria itself or by the cell as the result of infection with said mycobacteria, wherein the linkage is enzymatically cleaved by the enzymatic activity. In particularly preferred embodiments, the derivatizing group is a urea moiety that is specifically cleaved in Mycobacteria-infected cells by a mycobacteria-encoded urease.
The microparticle-drug conjugates of this invention have numerous advantages. First, the drug-microparticle conjugates are specifically taken up by phagocytic mammalian cells. Second, antibiotic, antimicrobial or antiviral compound, drugs or agents, most preferably anti-tuberculosis and anti-Mycobacterium compounds, drugs or agents comprising the drug-microparticle conjugates of the invention, are linked to the microparticle or covered by a coating comprising a specifically degradable moiety or material that is specifically cleaved upon entry into appropriate phagocytic cells, i.e., phagocytic cells infected with a tuberculosis-causing or other Mycobacterium-associated disease-causing microorganism. Third, the conjugates of the invention can be combined with other drug delivery approaches to further increase specificity and to take advantage of useful advances in the art. Fourth, the specificity of microparticle delivery to phagocytic cells and the specificity of conjugated antibiotic, antimicrobial or antiviral compound, drug or agent release in infected phagocytic cells permits the use and administration of efficacious antibiotic, antimicrobial or antiviral compounds, drugs or agents that are otherwise too toxic to be administered directly to an animal. Fifth, the specific delivery of the microparticles of the invention to phagocytic cells, and the specific release of antibiotic, antimicrobial or antiviral compounds, drugs or agents, and particularly activated embodiments thereof, permits direct administration of forms of said compounds, drugs and agents as they are activated by infectious organism-specific enzymatic or chemical modification, thereby providing a way of overcoming common forms of resistance to otherwise or previously efficacious antibiotic, antimicrobial or antiviral compounds, drugs or agents.
Thus, the invention also provides a method of killing a microorganism infecting a mammalian cell, preferably a phagocytic mammalian cell. This method comprises contacting an infected phagocytic mammalian cell with the compositions of matter or pharmaceutical compositions of the invention in vivo or in vitro. The invention also provides methods for treating microbial infections in an animal, most preferably a human wherein the infecting microbe is present inside a phagocytic cell in the human, the method comprising administering a therapeutically effective amount of the compositions of matter or pharmaceutical compositions of the invention to the human in a pharmaceutically acceptable carrier. Thus, the invention also provides pharmaceutical compositions comprising the compositions of matter of the invention in a pharmaceutically acceptable carrier. In most preferred embodiments, the infecting microorganism is a tuberculosis-causing microorganism such as M. tuberculosis, M. africanum or M. bovis. 
Thus, in a first aspect the invention provides compositions of matter, pharmaceutical compositions and methods for targeting antibiotic, antimicrobial or antiviral compounds, drugs and agents to phagocytic cells. In a second aspect, the invention provides compositions of matter, pharmaceutical compositions and methods for the specific release of antibiotic, antimicrobial or antiviral compounds, drugs and agents inside phagocytic cells. The invention in yet a third aspect provides compositions of matter, pharmaceutical compositions and methods for intracellular delivery of targeted antibiotic, antimicrobial or antiviral compounds, drugs and agents to phagocytic cells. In each of these aspects is provided compositions of matter, pharmaceutical compositions and methods for introducing antibiotic, antimicrobial or antiviral compounds, drugs and agents into phagocytic mammalian cells wherein the unconjugated compound, drug or agent would not otherwise enter said phagocytic cell, the compound, drug or agent would not be specifically targeted to said phagocytic cell or the compound, drug or agent would have deleterious or toxic effects on non-infected cells. In this aspect is included the introduction of said compounds, drugs or agents in antibiotic, antimicrobial or antiviral embodiments that would not otherwise enter the cell, for example, as charged embodiments or salts, or wherein the compound, drug or agent is unstable or has a short half-life. In addition, the antibiotic, antimicrobial or antiviral compounds, drugs and agents useful in this invention are provided in activated forms in which they are toxic to normal cells, or which are activated by infectious agent-specific enzymatic or chemical modifications, but which are conjugated to or coated within a microparticle of the invention and released only in phagocytic cells infected with a tuberculosis or other Mycobacterium-associated disease-causing microorganism In yet another aspect is provided compositions of matter, pharmaceutical compositions and methods for the specific coordinated targeting of more than one antibiotic, antimicrobial or antiviral compound to infected phagocytic mammalian cells. In another aspect, the invention provides compositions of matter, pharmaceutical compositions and methods for the introduction and specific release of antibiotic, antimicrobial or antiviral compounds, drugs or agents, preferably anti-tuberculosis and anti-Mycobacterium compounds, drugs or agents, and other compounds into cells infected by a tuberculosis-causing or other Mycobacterium-associated disease-causing pathological microorganism. In a final aspect, the invention provides compositions of matter, pharmaceutical compositions and methods for sequential, delayed, sustained or controlled intracellular release of antibiotic, antimicrobial, or antiviral compounds, drugs or agents impregnated within a coated, porous microparticle, or coated onto a nonporous microparticle, wherein the degradation of either a layer of the coating or the microparticle or both provides said sequential, delayed, sustained or controlled intracellular release of the antibiotic, antimicrobial or antiviral compounds, drugs or agents of the invention.
Specific preferred embodiments of the present invention will become evident from the following more detailed description of certain preferred embodiments and the claims.